Ecteinascidins have been identified, structurally characterized and synthetic methods for making them have been described. See for example, R. Sakai, et al., 1992, Prco. Natl. Acad. Sci. USA 89, pages 11456-11460, “Additional antitumor ecteinascidins from Caribbean tunicate: Crystal structures and activities in vivo”; R. Menchaca, et al., 2003, J. Org. Chem. 68(23), pages 8859-8866, “Synthesis of natural ecteinascidins (ET-729, ET-745, ET-759B, ET-736, ET-637, ET-594) from cyanosafracin B”; and I. Manzanares, et al., 2001, Curr. Med. Chem.—Anti-Cancer Agents, 1, pages 257-276, “Advances in the Chemistry and Pharmacology of Ecteinascidins, A Promising New Class of Anticancer Agents”; and references therein. These references describe ecteinascidins. Examples of ecteinascidins are provided by ET-743, ET-729, ET-745, ET-759A, ET-759B, ET-759C, ET-770, ET-815, ET-731, ET-745B, ET-722, ET-736, ET-738, ET-808, ET-752, ET-594, ET-552, ET-637, ET-652, ET-583, ET-597, ET-596, ET-639, ET-641, and derivatives thereof, such as acetylated forms, formylated forms, methylated forms, and oxide forms, such as N-oxide forms.
The structural characterizations of such ecteinascidins are not given again explicitly herein because from the detailed description provided in such references and citations therein; any person of ordinary skill in this technology is capable of obtaining such information directly from the sources cited here and related sources.
At least one of the ecteinascidin compounds, ET-743, has been extensively studied, and it will be referred to specifically herein to illustrate features of this invention.
Ecteinascidin 743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the marine tunicate Ecteinascidia turbinata and has the following structure:

A pharmaceutical composition comprising ET-743 in combination with a pharmaceutically acceptable carrier, diluent or excipient is claimed in U.S. Pat. No. 5,256,663.
A recent review of ET-743, its chemistry, mechanism of action and preclinical and clinical development can be found in van Kesteren, Ch. et al., 2003, Anti-Cancer Drugs, 14 (7), pages 487-502: “Yondelis (trabectedin, ET-743): the development of an anticancer agent of marine origin”, and references therein.
ET-743 possesses potent antineoplastic activity against a variety of human tumour xenografts grown in athymic mice, including melanoma and ovarian and breast carcinoma.
In clinical phase I studies of ET-743, promising responses were observed in patients with sarcoma and breast and ovarian carcinoma. Therefore this new drug is currently under intense investigation in several phase II clinical trials in cancer patients with a variety of neoplastic diseases.
As it is explained in WO 0069441, incorporated in full by reference, ET-743 is supplied and stored as a sterile lyophilised product, having ET-743, mannitol and a phosphate buffer. A preferred formulation is one obtained from 0.9% sodium chloride or other suitable infusion vehicle, 250 μg of ET-743 with 250 mg of mannitol, 34 mg of monopotassium phosphate, and phosphoric acid to adjust the pH. This formulation is then reconstituted and diluted for intravenous injection.
ET-743 is a complex chemical entity, as revealed by its structural features. In addition, ET-743 exhibits limited aqueous solubility, and its stability, particularly in biocompatible forms and formulations, is difficult to predict and achieve. These characteristics challenge the ordinary skills and conventional methodologies in this technology, particularly when it comes to the preparation of ET-743 formulations that are to be readily used for medical purposes. Such uses preferably rely on formulations whose characteristics include one or more of the following: biocompatibility, stability under ambient conditions, or under conditions that are as near to ambient conditions as possible, with a shelf life that is as long as possible, and easy reconstitutability to form reconstituted solutions that are as stable under ambient, or near ambient conditions, for as long as possible.
However, conventional formulations and methodologies for preparing such formulations do not provide desirable features and characteristics such as those referred to above. For example, the cited review of 2003 by van Kesteren Ch. et al. reports that
ET 743 has limited aqueous solubility. However, by adjustment of the pH to 4, adequate concentrations of ET 743 could be reached. Instability of ET 743 in aqueous solution necessitated lyophilization in order to increase the storage stability of the pharmaceutical product. ET-743 is currently formulated as a sterile lyophilized product containing 250 μg active substance per dosage unit, 250 mg mannitol as a bulking agent and 0.05 M phosphate buffer at pH 4 in order to solubilize ET-743. This formulation is unstable with long-term storage at refrigerated and room temperature, and should therefore be stored between −15 and −25° C., protected from light. Reconstitution is performed by adding 5 ml Water for Injection, with subsequent dilution in normal saline before i.v. infusion. The reconstituted solution is stable at ambient temperature for up to 24 h.
In practice this product containing 250 μg of ET-743 is manufactured by freeze-drying 5 ml of solution containing ET-743, mannitol, phosphate buffer and water in a moulded vial. Moulded vials containing 1 mg ET-743 are also manufactured by freeze-drying 20 ml of the solution.
Freeze-drying typically involves freezing the solution, reducing the pressure for a period of primary drying to remove water vapour from the frozen material by sublimation and give a semi-dried mass, and increasing the temperature for a period of secondary drying to remove residual water from the semi-dried mass. The vials are then sealed.
The above-described conventional ET-743 formulation suffers from several disadvantages. One of them is that the lyophilised ET-743 formulation has to be stored at about −20° C. to prevent decomposition of the ET-743 in order to achieve a shelf life of at least 18 months.
In addition, ET-743 formulations face the problem of formation of relatively large amounts of ET-701 as impurity. ET-701 is the main impurity produced during the lyophilisation process and during storage of the ET-743 formulation. It comes from the hydrolysis of ET-743 and has the following structure:
Formation of impurities, however, diminishes or even forestalls the ability to standardize formulations. It is consequently desirable to provide formulations and methods for making the same that provide embodiments whose composition does not readily and unpredictably change by the uncontrolled formation of impurities.
Furthermore, another disadvantage of the above-described conventional ET-743 formulation methodology is that in order to obtain the lyophilised formulation it is necessary to freeze-dry a relatively large amount of solution with fill volumes in the order of 5 to 20 ml. In contrast, it would be desirable to develop a manufacturing methodology for formulations with compounds as complex as ET-743 that permits the making of formulations with higher active substance concentrations, so that the volumes to be handled are consequently reduced. Time and energy are needed in conventional methods for the step of freeze-drying, in view of the relatively high fill volumes of 5 or 20 ml. Along with the time and energy, there is also the risk of decomposition of the ET-743, particularly in the secondary drying.
In view of the potential of ET-743 formulations as antitumoral agents, there is a need to provide a formulation that can solve problems that conventional formulations and manufacturing methodologies do not address or do not completely solve. These problems include the problem of stability of ET-743. Embodiments of ET-743 formulations should preferably exhibit favourable freeze-drying properties, should preferably be susceptible of ready reconstitution, and they should preferably exhibit dilution properties, such as upon dilution with infusion fluid, while presenting as many as the desirable characteristics of formulations for medical use as referred to herein. As indicated above, embodiments of ET-743 formulations should be stable during long term storage. In addition, the formulation and its manufacturing methodology should satisfy biocompatibility standards and should thus allow for the effective use of a formulation vehicle that is non-toxic, at least at the concentrations used for infusion.
A general review of excipient-drug interactions in parental formulations is provided by Akers, M J, in Journal of Pharmaceutical Sciences, 91, 2002, 2283-2300. This reference provides, inter alia, a section on bulking agents and lyoprotectants, including this subject matter in the context of lyophilisation.
It is envisaged that the methodologies and formulations developed in the context of this invention are applicable to other ecteinascidins, in addition to ET-743.